Despite the remarkable progress made in deciphering embryonic stem cell pluripotency, particularly in murine models, there is a fundamental gap in understanding how human embryonic stem cells (hESCs) regulate the pluripotent state. Our long-term goal is to decipher the architecture of the regulatory network that allows for un- restricted hESC proliferation while preserving their potential to form the full repertoire of cell types found in the human body. To define the composition and function of this network, we first searched for transcriptional regulators of hESCs through shRNA-based functional screen. Several factors were identified that are required for the maintenance of the pluripotent state including three novel genes, BCOR, ZFP42 and ZNF649, that function to repress hESC differentiation. While the requirements for ZFP42 and ZNF649 varied among different hESC lines, depletion of BCOR resulted in rapid differentiation in all cell lines tested indicating that BCOR is a core component of the primed pluripotent state. As only a few core factors have been identified, studying BCOR is a high priority. The objectives in this application are (1) to elucidate the mechanistic aspects of differentiation repression by BCOR and (2) to identify-specific hESC regulatory mechanisms genome-wide. Our central hypothesis, formulated based on the preliminary data and prior work on BCOR in other cell systems, is that BCOR suppresses differentiation of hESCs by recruiting KDM2B and RING1A/B modules to generate repressive chromatin footprints at the target sites. This hypothesis will be tested through the following specific aims: 1) Determine how the BCOR complex represses its target genes in hESCs and 2) Determine how the BCOR complex is recruited to target genes. In addition, we propose to 3) comprehensively identify regulatory path- ways that modulate pluripotency in hESCs. Under the first aim, biochemical, functional and genomics approaches will be combined to determine (1) BCOR target genes and the nature of the BCOR complex(es) in hESCs; (2) relative contributions of KDM2B and RING1A/B to the complex function; and (3) the mechanism that maintains unique epigenetic signatures at BCOR target sites. Under the second aim, the BCOR complex will be purified from hESCs and sequenced using mass spectrometry in order to define DNA-/ chromatin binding proteins that physically interact with BCOR which then will be tested for their ability to recruit the complex to targets. In addition, recruitment via the canonical PRC-dependent mechanism and via long non-coding RNAs will be investigated. Under the third aim, positive and negative regulators of hESC pluripotency will be identified using an unbiased whole-genome shRNA screen. Our approach is innovative, because it utilizes novel state-of-art technologies to obtain new insights into the regulatory complexity of hESCs. The proposed re- search is significant, because it is expected to vertically advance and expand understanding of how hESCs cells control the pluripotent state. Such knowledge has the potential to enhance hESC maintenance and differentiation - critical steps for new and innovative approaches to treatment of a variety of diseases.